THE TOXICITY OF POTASSIUM CYANIDE TO TROUT

THE TOXICITY OF POTASSIUM CYANIDE TO TROUT

BY D. W. M. HERBERT AND J. C. MERKENSThe Water Pollution Research Laboratory, Watford, Herts

(Received 6 March 1952)

(With Five Text-figures)

INTRODUCTION

Lack of fundamental information on the factors determining the survival of fishin water containing poisonous substances has led to the use of a variety of experi-mental techniques and methods of interpreting results in studies on the toxicity tofish of constituents of waste waters discharged to streams; information on thesurvival times of fish in solutions where they survive for longer than one or two daysis very scanty, and a programme of laboratory studies on the survival of rainbowtrout in solutions of cyanide has therefore been started with the object of providingbackground knowledge for studies of this kind.

In nearly all the toxicity tests reported in the literature, fish have been immersedin a fixed volume of poisoned water, but both Allee & Bowen (1932), using a sus-pension of colloidal silver, and Carpenter (1927), using solutions of lead nitrate,have shown that when fish are kept in the test solution the concentration of poisonfalls and survival time can be increased by increasing the number of fish in thesolution. From their experiments these workers concluded that the reduction inconcentration of poison was partly due to precipitation by mucus. It is probablethat similar reactions reduce the concentration of many lethal agents and this,together with other changes produced in the water by the metabolism of the fish,would result in a continually changing environment during a test with a fixedvolume of solution, so that the survival times could not be assumed to be repre-sentative of those of fish in a continuously polluted river. The simplest way toovercome this difficulty is to test the fish in a vessel through which a stream ofpoisoned water flows at such a rate that the activity of the fish cannot seriously alterits composition. Such an apparatus has been used by Erichsen Jones (1938), butit was too small for more than two minnows to be tested at one time. It is desirableto test more fish than this in one concentration if variations in resistance of fish toa poison are to be assessed. It was therefore decided to make tests in a vessel inwhich fifty yearling rainbow trout could be tested together in a stream of water inwhich concentration of poison, dissolved oxygen tension, and temperature werekept as constant as possible.

MATERIALS AND METHODSApparatus

A constant concentration of poison is maintained by adding a solution of poisonat a constant rate (approximately 2 ml. a minute), to a stream of dechlorinated andoxygenated tap water flowing at a constant rate of about 2 1. a minute, mixing the

The toxicity of potassium cyanide to trout 633

two streams thoroughly, and passing the poisoned stream through a test tankholding 200 1., in which the water is thermostatically controlled to the requiredtemperature. Fig. 1 is a flow diagram of the apparatus. Mains water is passedthrough a sand filter to remove suspended solids and then through a tower filledwith activated carbon to remove any chlorine. The treated water is oxygenated bypumping at high velocity through a glass filter pump into a storage tank. From

Valve (

Constant delivery dosing pumpCentrifugal \~~1

pump ^ '

Mixing !~ ~ ~

Ii — - -"a

Them

dnosut—

Delay unk Test tank

Immersionheaters

— — Storage tank

Fig. i. Flow diagram of the apparatus used to test fish in a stream of water containing a constantconcentration of poison at a controlled temperature. (Not to scale.)

the storage tank the water is pumped up to a constant level tank which providesa constant head above an orifice plate in the outlet pipe and controls the flow ofwater to a constant rate close to 2 1. a minute.

Water from the constant level tank falls into the receiver of the mixing vesselinto which a solution of poison is added at a known rate by a constant deliverydosing pump. Thorough mixing is effected by passing the water and the solutionof poison through small holes in the central pipe of the mixing vessel. From themixing vessel the water flows through a throttle to a delay tank of 50 1. capacity, inwhich any small fluctuations in concentration of poison are smoothed out, and fromthere it flows into the heavily glazed iron test tank. The inlet pipe forks into twobranches which run along the base of the tank close to each of the long walls, andthe poisoned water enters the tank through small holes in the walls of the pipes.The water in the tank is heated by two 2 kW. immersion heaters, heavily platedwith tin, and the temperature is controlled by a thermostat of the bimetal spiraltype enclosed in a tinned metal pocket projecting into the water through the sideof the tank.

634 D. W. M. HERBERT AND J. C. MERKENS

PERFORMANCE OF THE APPARATUSMaintenance of constant concentration of poison

The rate of flow of water through the mixing vessel will remain within ± 0-33%of the mean rate for many weeks, and the variations in delivery rate of the dosingpump rarely exceed ± 1 -4 % of the mean rate. In one test a solution of Rhodamine B,a fast dye which can be determined photoelectrically in concentrations of abouti-o p.p.m. to within ±2-32%, was added to give i-o p.p.m. in the test tank; theresults of nine determinations made over a period of 21 hr. lay within ±2-78%of a mean value of 0-99 p.p.m.

Maintenance of the required concentration of cyanide in the test tank is oftencomplicated by an apparent loss of cyanide when this is determined by analysis.The amount lost varies with different samples of water, and the cause of thiscyanide demand is not known; it is usually observed with the tap water used in thislaboratory and with distilled water. All cyanide determinations reported in thispaper were made by a modification of Robbie's (1944) phenolphthalein method(Merkens, in preparation). With this modification, concentrations of cyanidebetween 0-2 and 0-03 part cyanide per million can be determined within approxi-mately ± 4 % .

Dissolved oxygen concentration

Measurements were made over a 3-day period and at a temperature of 150 C ,of the dissolved oxygen tension in water entering and leaving the test tank; fiftysmall rainbow trout were in the tank throughout. The dissolved oxygen in theinflowing water varied from 100 to 105% of saturation (mean 102-7%) a n d in theoutlet from the test tank it varied from 93 to 101 % of saturation (mean 95-9%).

Temperature control

The temperature in the test tank can be controlled to within ± 0-5° C. Exceptwhere otherwise stated, all experiments reported in this paper were made ati7-5 ± o-5° C.

pH valueNo pH control is incorporated in the apparatus as at present used; the pH value

of the water varies between 7-4 and 8-o. Wuhrmann & Woker (1948) have shownthat the toxicity of cyanide to fish is affected by the proportion of the cyanide ionto the undissociated molecule, HCN being more toxic than the equivalent concen-tration of hydrogen and cyanide ions. According to Wuhrmann & Woker about97 to 93 % of the hydrogen cyanide produced by hydrolysis of the added KCNwill be in the undissociated condition in a pure solution between pH values of7-4 and 8-o. The presence of other ions in the solution will modify these proportions,but random fluctuations of pH value within these limits should not greatly alterthe toxicity of cyanide.

Test animalsAll the experiments reported were made with rainbow trout between -9 months

and 2 years old, obtained from a commercial trout farm. The fish were kept in thelaboratory in aquaria supplied with running, continuously aerated, dechlorinated


tap water; the temperature of the water was not controlled. The fish were fed oncedaily on finely minced bullock's heart.

Before every test in cyanide solution the fish to be used were removed from theirstock aquarium to another which was maintained at the temperature of the experi-ment (i7'5° C). Except in Exp. 4, they were not fed from the time they enteredthe acclimatization tank until the end of the experiment.

In all tests which were completed in 9 hr. or less (that is in concentrations of0-14 part cyanide per million or more) the fish were watched continuously; in testsof longer duration they were observed only at intervals, but the interval was nevergreater than 1 hr. except for the last surviving fish in the test in 0-09 part CN permillion in Exp. 3. In those experiments where the fish were continually observed,the time from immersion in the poisoned water until each fish had overturned andremained motionless on its back or side for 5 sec. was recorded. Experience hasshown that after a fish has remained overturned for 5 sec. it rarely regains equi-librium while remaining in the poison, unless disturbed. Fish which had over-turned were taken from the test tank and placed in well-oxygenated unpoisonedwater. Fish removed immediately usually regained equilibrium in a few minutesand showed no ill effects from the poison, but in the experiments where the fishwere observed at intervals they were often dead when first seen to be affected, andthose which were overturned but still alive did not always recover when transferredto unpoisoned water. In these cases the survival time was taken as the time inminutes from the beginning of the experiment until the time when the fish wasfirst seen to be overturned or dead.

EXPERIMENTALSelection of acclimatization period

To reduce errors caused by variations in environment, conditions, such as tempera-ture, in the test tank were standardized as far as possible. To keep the temperaturein the test tank constant throughout a long series of tests, the experimental tempera-ture has to be above that of the mains water supplying the aquaria, so if fish weretransferred direct from aquaria to test tank they would be subjected to abrupttemperature changes. Sumner & Wells (1935) have shown that the resistance offish to cyanide and some other poisons at one temperature is affected by the dif-ference between the temperature of the aquaria in which they have been living andthe test solution. This source of variation could be avoided by keeping all aquariaat a constant temperature, but since facilities for this are not available the fish wereacclimatized for a period at the experimental temperature before each test. InExp. 1 the effect was studied of different periods of acclimatization on the over-turning time of rainbow trout in cyanide, so that a suitable period for acclimatizationcould be chosen.

Eighty young rainbow trout (mean length 898 cm.; standard deviation 0-94 cm.)were graded for size, and sorted into two batches as nearly as possible equivalentwith respect to length. The tank used for acclimatization was divided by a partitionperforated with holes through which fish could not pass. A batch of fish was kept


in each hah0 of the tank, so both were subject to similar environmental conditions.Batch A was immersed in 0-15 part CN per million after spending 24 hr. in theacclimatization tank. When each fish overturned the period of survival beforeoverturning was recorded and it was replaced in the acclimatization tank. Thisbatch remained in the acclimatization tank until the last day of the experiment whenit was tested again. Batch B was tested in the same way after acclimatization for48 hr. and again on three subsequent days. The fish were returned to the acclimatiza-tion tank after each test. During the experiment five fish were lost from batch Aand two from batch B.

Results of this experiment are summarized in Table 1.

Table 1. Effect on resistance of rainbow trout to potassium cyanide of increasingtime of acclimatization to the temperature of the tests (17-5° C.)

Batch of fishtested

ABBBBA

Period ofretention in

acclimatizationtank beforeimmersion in

cyanidesolution (hr.) j

No. of fishavailablefor test

97120172191 I

364040403835

Cyanideconcentration

required (partsCN per million)

Mean of cyanideconcentrations

obtained byanalysis

(parts CN permillion)

Mean periodof survival

(min.)

0-1500-1500-1500-1500-1500-150

0-1500-1510-1490-1490-1500-150

28-81286035-15469739-6850-80

Survival time increases with the duration of the acclimatization period. A regres-sion analysis (Emmens, 1948) shows that the increase is highly significant statis-tically, while the deviations from the best fitting straight line are not significantat the 0-02 probability level. There is no evidence of a difference between batches Aand B, so it is rather unlikely that the increasing resistance of the fish to the cyanidewas due to repeated exposure to it. It is possible that if the trout had been acclima-tized for longer periods their resistance to cyanide would have reached a steadystate, but evidently more than 8 days are required for this. Practical considerationsnecessitated an acclimatization time of not more than a few days, and since itappears from Exp. 1 that differences in the duration of acclimatization wouldproduce varying resistance to cyanide, a 2-day period of acclimatization was adoptedas standard practice, and in the experiments that follow all tests were started asnearly as possible 48 hr. after the fish were put into the acclimatization aquarium.

Length of fish and resistance to cyanide

Yearling rainbow trout vary considerably in size even when they have all beenreared under similar conditions. Exp. 2 was made to determine whether resistanceto cyanide was correlated with body length.

All the fish used were taken from one pond at the trout farm. The supplier saidthey were all of the same age and had been reared under the same conditions since


they were spawned. Sixty-three 1-year-old rainbow trout ranging in length from5-5 to 17*25 cm. were kept without food in running water at 17-5° C. for 48 hr.before the experiment, and were then immersed together in the test tank throughwhich flowed a stream of water to which potassium cyanide was added to bring theconcentration to the equivalent of 0-16 part CN per million. The cyanide demandof the water reduced this to an average value of o-15 3 part CN per million (maximum0-154 P-P-in., minimum 0-152 p.p.m.). The periods of survival of fish of differentlengths are shown in Table 2. There is an obvious tendency for the longer fish tosuccumb sooner than the shorter fish.

Table 2. Relation between length of yearling rainbow trout and periodof survival in potassium cyanide

(Length of each fish measured to nearest 0-25 cm.)

Range of length(cm.)

5-5- 6-256 5 - 7257-5- 8-258-5- 9259-5-10-25

10-5-11-2513-5-14-2514-5-15-2515-5-16-2516-5-17-25

No. ingroup

311

710

831

1361

Mean length(cm.)

5757 - 1 37-938969-81

11-co14-0015-00I5-7I17-25

Mean periodof survival

(min.)

39O37-o33-424-1222528-3I2-O18-41 6 716-0

This relationship between the length of a rainbow trout and its survival time insolutions of cyanide is important in the design of toxicity tests, because if the fishto be used in an experiment are selected so that the range of size covered is small,the variance of overturning time will be reduced and the error of statistics such asmean period of survival and median lethal time will be smaller. At the same time,however, the statistics will be less generally representative of the fish of the agegroup tested. It would be worth while using fish of as nearly as possible the samesize to increase the sensitivity of an experiment where the effect of dissimilartreatment was to be compared, but not where the results will be required to showthe range of variation typical of a more natural population.

By doing separate tests on various length groups, or by recording the length withthe survival time of each fish, results typical of a wider range of length and givingaccurate comparison between groups could be obtained.

Distribution of resistance to cyanide and the relation betweencyanide concentration and survival time

Exp. 3 was designed to investigate the relation between concentration andsurvival time over the range 2-0 parts to 0-07 part CN per million and to provideinformation from which the distribution of resistance to the cyanide concentrationsused could be deduced.


Material and experimental design

It was not possible to obtain a large enough stock of fish representative of therange of length typical of one age group without drawing them from differentsources and entailing the risk that differences due to environment would be con-founded with those due to length. We therefore decided to obtain all fish from onesource and to restrict the range of length as much as possible.

Rainbow trout, about 9 months old and approximately equal in size, wereselected from one pond at the trout farm where they had been reared together; theirmean length was 9-52 cm. (standard deviation 0-98 cm.). The fish were groupedinto five classes according to length, and each class was sorted at random intotwenty aquaria where the fish were kept until used in the experiment. In this waythe stock was divided into twenty samples nearly equivalent with respect to lengthand with all properties other than those associated with length distributed atrandom. The fish were grouped in this way to make the batches more alike inresistance to cyanide, but it causes the significance of the differences betweenbatches to be underestimated to a slight extent.

All batches were acclimatized to the experimental temperature (17-5 ± 0-5° C.)for.48 hr. before the start of each test and were not fed until the end of the test.The experimental design provided for twelve tests in concentrations between2*0 parts and 0-07 part CN per million and one control in unpoisoned water.The other seven batches were held in reserve to be used if a test batch had to bediscarded for any reason. The batches were used and the thirteen concentrationswere tested in random order. For most tests the fish were immersed together inthe test tank and the time of survival of each fish was recorded, but in the test ini-o part CN per million the fish overturned so rapidly that not all the survival timescould be recorded. When the tests were performed in 2-0 parts and 0-3 part CNper million, the fish were immersed five at a time.

The concentrations of cyanide used, and the values obtained by analysis duringeach test are summarized in Table 3.

About halfway through the series of tests one batch of fish was immersed in astream of unpoisoned water at 17-5 ± 0-5° C.; they were not fed. The first fish diedafter 30 days and 50% had died after 68 days. Since no test in cyanide lasted morethan a fortnight, no compensations for natural mortality were made.

Distribution of resistance to cyanide and the relation between themeans and variances of survival times

When the percentage of fish overturned in any one of the concentrations ofcyanide tested is plotted against time, the points lie approximately on a sigmoidcurve. Bliss (1937) has shown that such sigmoid time-mortality curves can bedescribed by assuming that the individual survival times or functions of these timessuch as the logarithms or reciprocals are normally distributed. The examination ofthe data of this experiment by the usual statistical techniques and tests would bestrictly valid only if the survival times, or some functions of the survival times,


satisfied two criteria; namely, the distribution of the times or transformed timesshould be normal within each concentration, and the variances of the distributionsin the different concentrations should be equal after allowing for the variation dueto random sampling.

Table 3. Concentrations of cyanide added in Exp. 3 and determined byanalysis in the test tank

Concentrationi M l l H \ I I LI 0 L*&^^*1

added ^DartsCN per million)

2 - oi - o0 - 30 2 5O-2O0 1 8

o-i60-14o-io0-090 0 8

0-07

Mean of concen-trations obtainedby analysis (partsCN per million)

1-9660-9650-2930-2260-1970-1600-1600-1320-08460-07360-07190-0598

|

No. ofCyanide lost , determinations

(p.p.m.) made duringtest

0-034 30-035 20-007 1 3O-O24 2

0-003 3O-O2O 8o-ooo 50-008 70-0154 , 740-0164 610-0081 70O-OI02 j 132

Coefficient ofvariation of

concentrationsobtained by

analysis

2 3 9o-731 6 72 5 20 7 8o-531 1 31-493-589-18*i-952-98

• Large coefficient of variation probably caused by unusually large fluctuations in cyanide demandof the water.

There are two ways of examining the first criterion. In the first place, probitcurves can be drawn. In Fig. za the survival times recorded during the test in0-14 part CN per million are plotted as a sigmoid time-mortality curve, and inFig. 26 the probits corresponding to the percentage of fish overturned are plottedagainst time, log time, and i/time. If overturning times were normally distributed,the points in the probit-time graph would lie on a straight line, but since the plotsof both times and 1 /times against probits are curved, and the logarithms of timesalone are reasonably linear, it can be concluded that log-survival times are bestfitted by the normal curve. The second method is to examine the goodness of fitmathematically; this has been done for each concentration, except i-o part CNper million where insufficient individual times were recorded, and the results aresummarized in Table 4. The tests employed were the •>//}1 and a tests for skewnessand kurtosis (Geary & Pearson, 1938). At the 5% level of significance the survivaltimes and their reciprocals were consistent with the assumption of normality inonly one and three concentrations respectively. The logarithmic transform departssignificantly from normality in only five of the eleven concentrations.

As the concentration of cyanide decreases and the survival times of the fishincrease, the range of individual survival times also increases. This is shown inTable 5, columns 2 and 3, where the variance of survival times increases with meansurvival time. The reciprocals of times show a similar trend, but it is not apparentwith the logarithms of time for concentrations less than 0-25 part per million. The

I K B.2IJ, 4 41

640 D . W. M. H E R B E R T AND J. C. M E R K E N S

last nine variances in column 5, Table 5, have been shown by Bartlett's test to besignificantly non-homogeneous, that is, they cannot be assumed to be estimates of

25

SO

75

100

V\•

a

- — _80 120Survival time (mln.)

160 200

i

1-5Log (survival time, mln.)1-25 2-0 2-25

N

510

25

50

75J

9 0 '

95

100 200 30 20 10 0Survival time (mln.) 100/survlval time (mln.)

Fig. 2. Survival times of rainbow trout in a solution containing C14 part CN per million at i7-s°C.(a) Percentage survivors plotted against time. (6) Probits corresponding to percentage survivorsplotted against time, log time, and rate.

a single population variance of log times common to all the tests. Since they appearto vary at random, however, it is probable that this heterogeneity was caused byvariations in conditions not under experimental control.

The toxicity of potassium, cyanide to trout 641

Table 4. The fit of normal distribution curves to overturning times, logoverturning times, and reciprocals of overturning times

Concentration(parts CN

per million)

2 - 0

o-30 2 5O-2OO l 80-160-14o-io0-090-080-07

No. of casesboth tests

N

46463 136484742474 0

454 1

passing

Time

A- 1 - 2 *

o-6*i-6*i-8*I O *O-2I O *

o-6*3 6 *0-3I - I *

a

0 7 80-830-69*0-72*0-770-770 7 90-73*0-58*0-76*o-43*

1

Log

A- 2 - 5 *

o - iI I *

0-9*- 0 - 5— o-i

o-o- i - 8 *

0-5- 1 - 2 *- 0 - 3

time

a

0-69*0-820-780-78o-750-760-790-67*0-770-74*0-78

6

i/time

A4-2*O-I

- 0 - 3O-2

i-8*2-S*1-a*4'4#

1-4*a-9*1-9*

a

018*o-8i0-780 7 80-69*o-63*0-74*0-49*O-74*0-63*0-71*

3

N= number of individuals in test. A znd a = estimates of Vft e

In a normal distribution Vft=° , and 0 = 0-79788.• Departures from normality significant at 5 % probability level.

a (Geary & Pearson, 1938).

Table 5. Means and variances of survival times, logarithms and reciprocals ofthe survival times of rainbow trout in various concentrations of cyanide

Cyanideconcen-tration

(p.p.m.)( 1 )

2-O0 - 30-25O-2OO l 80-160-14o-io0-090-080-07

Mean ofsurvival

times(min.)

( 2 )

2-668-84

12-4112-1224-8672-4OOO-2O

2,523-131,617-503,600-514,44i

Variance ofsurvival times

(3)

0-0183-3°

30-4637-79

1.6553.O59i,749

1,482,9001,174,6101,649,1003,996,400

Meanof log

survivaltimes

(4)

0-4178093761-06271-04221-55041-74821-91153-33O83-I5O73-52423-5675

Varianceof log

survivaltimes

(5)

0-00690-00760-02390-03190-06160-10040-03840-08630-04480-03220-0337

Mean ofreciprocals ofsurvival times

(6)

0-39060-1178

i o - 1 x 0-9151io"1 x 0-9780io-'x 0-3389io~xx 0-2352i o - 1 x 0-1391io-'x 0-6679io"'x 0-7902i o - ' x 0-3310io-'x 0-2555

Variance ofreciprocals ofsurvival times

(7)

i o - ' x 0-1040io-'x 0-5616io"*x 0-772910""* x 0-1240io-'x 0-566610-* x 0-4369io"4 x 0-370110-* x 0-9764io"*x 0-1498io"'x 0-3604io-'x 0-1644

The joint evidence of the two criteria is that the log transform is markedlysuperior to the other two forms, but since even the log times depart from thesymmetrical homogeneous form more than would be expected from randomsampling, the hypothesis of log normality with uniform variance cannot be un-questionably accepted. It is known, however, that the departures from the twoideal conditions already stated have to be considerable before conclusions drawnfrom the statistical analysis are invalidated. It is extremely unlikely that thedeparture of the log transform from the normal homogeneous form is sufficientlyserious to vitiate the inferences to be drawn from the analysis, as all are shownto exist at a very high level of statistical significance.

41-2


Relation between concentration of poison and survival time

Where the logarithms of the survival times of fish immersed in a solution ofpoison are normally distributed the best estimate of the median survival time, ortime taken to overturn 50% of the test fish, is given by the geometric mean of thesurvival times

Median survival time = antilog — ^ — , (1)

where T is the survival time of each fish, and N is the number of fish in the test.In Fig. 3 the median survival times estimated from each test by equation (1)

are plotted against the concentrations of cyanide added to the test tank, in bothcases on logarithmic scales. If the mean concentrations of cyanide obtained byanalysis are plotted instead of the concentrations of cyanide added a very similarcurve is obtained but the position and slope are slightly different. Over most ofthe curve, logarithms of concentration and logarithms of survival time are linearlyrelated by the line A-A1, but at the higher concentrations the points divergesystematically from it.

The equation for the line A-A1 is

nlogC+log T=logk, (2)

or CnT=k, (2a)

where C is concentration of cyanide in parts per million; T is survival time inminutes; and n and k are constants.

A regression analysis has been carried out for the data corresponding to concen-trations less than 0-2 p.p.m.; the constants of the best fitting straight line aren = 5-639 and log k= — 2-700. Both the slope of the line and the departures of thegroup means from the line are highly significant statistically (P < o-1 % in each case).Since the group means are scattered about the line at random over this region, withno systematic trend away from it, equation (2) can be assumed to represent therelation between C and T, and the results of the regression analysis to show thaterrors in the estimation of T are mainly due to factors acting at random whichincrease or decrease the survival times of all the fish in a batch, while the errorsdue to the variation of survival times within a batch are relatively insignificant.Inaccuracies in the maintenance of the required concentration of cyanide couldaccount for some of this error, and factors associated with differences in environmentbetween the stock aquaria, the different dates on which the tests were started, oruncontrolled variations of the chemical and physical properties of the water affectingthe response of the fish might also contribute to it.

Because deviations of group means from regression constitute the major sourceof error in the experiment, the significance of such departure from the line as isshown by the highest concentrations must be assessed from the scatter of mediansurvival times and not from the deviations of individual log survival times from thegroup means. The dotted lines at either side of A-A1 (Fig. 3) represent the limitswithin which 95 % of the median survival times should lie over the range of con-


centrations for which equation (2) holds. They have been calculated on the assump-tion that the constants of the line are known without error. To avoid including pointsnear the region where A-A1 meets B-B1 the five shortest median survival times werenot used when calculating these limits. All points except those for concentrations

10,0001

005 0-1 0-2 05 10 2-3Cyanide concentration In parts CN per million (logarithmic scale)

Fig. 3. Relation between concentration of cyanide and survival time of rainbow trout at 175° C.The points represent the median survival times and the dotted lines 95 % confidence limitscalculated from the scatter of the seven longest median survival times about the fitted line.A-Al and B-B1, for explanation see text.

of 0-3 part CN per million and more lie within the band; there is therefore nosignificant departure from equation (2) between 0-25 and 0-07 part CN per million,and the intersection of A-A1 and B-B1 presumably occurs between 0-25 and 0-3part CN per million.


Factors associated with the varying resistances of trout to cyanide

The differing resistances of individual rainbow trout to cyanide at the time of anyone test has been shown in Exp. 2 to be due in part to a relation between lengths andresistance but some could be due to other inherent and permanent differencesbetween individuals, to short-lived random effects of the environment, or to acombination of both these factors. In Exp. 4, which was designed to estimate therelative importance of these possibilities, use was made of the rapid recovery oftrout from cyanide poisoning if they are returned to unpoisoned water shortly afteroverturning.

Method

Fifty rainbow trout were distinguished by cutting different combinations ofnotches in the borders of the fins; after marking they were kept in an acclimatizationaquarium at 17-5° C. until the end of the experiment. They were not fed duringa period of 48 hr. before each test. Eight tests were made over a 3-week period.In each test the fish were immersed in a potassium cyanide solution containing0-15 part CN per million and the overturning time and markings of each fish wererecorded. Losses of fish from a variety of causes during tests 1-7 reduced thenumber of fish to thirty-eight and another loss reduced the number available intest 8 to twenty-six. When the experiment was completed it was found that therewas no significant correlation between the overturning times of the trout and thenumber of notches cut, so presumably this method of marking does not affect theresistance of fish to cyanide.

Results

There was an obvious tendency for any one fish to retain its original position inthe overturning order throughout the series of tests, although the order was neverexactly the same on any two occasions. The results of the experiment are summarizedin Table 6. The correspondence between the survival times of individuals duringthe series has been assessed by calculating the product moment correlation coeffi-cients (r) between the overturning times in test 1 and the times in tests 2-8; inevery case the correlation is very significant.

Since there was not an absolute correspondence between the overturning timesin the eight tests, the resistance of each individual cannot be due entirely topermanently inherent factors, but the stable and significant correlation betweenoverturning times shows that such factors, persisting for at least 3 weeks, have arole in determining the survival time of an individual; the actual overturning timeis the resultant of permanent, intrinsic factors and short-lived effects of the environ-ment. It can be shown by the reasoning in Peters & Van Voorhis (1940, p. 120et seq.) that the correlation coefficients in this experiment have the meaning

r =


where <r| is the variance due to the intrinsic causes persisting in the fish throughthe series of tests, and cr| is the variance due to the other factors which havemodified the inherent resistance. The proportion of the variance of survival timedue to the intrinsic factors in this experiment is between 66-14 and 84-28%.

Table 6. Summary of results of experiment to determine the extent to which thevarying resistances of rainbow trout to potassium cyanide are due to inherent andpermanent differences between individual fish

Test no.

1234

6

8

Time fromstart of1st test(hr.)

3*2448i5172

1925°7i

Cyanide concentration

Desired(parts CN per

million)

0-1500-1500-1500-1500-1500-1500-1500-150

Mean valueby analysis

(parts CN permillion)

0-152O-I550-1490-1500-1510-1500-140O-I57

Meanperiod ofsurvival(min.)

199318642456299025-72283043-O35I-76

Correlation coeffi-cients between theoverturning times

Betweentests

1 and 21 and 31 and 41 and 51 and 61 and 71 and 8

T

0-840840-740-740-78o-660-71

N

38383838383826

r=product moment correlation coefficient; N= number of pairs of observations used in calcu-lating r.

DISCUSSIONThe method usually employed to interpret the results of toxicity studies with fishderives from that used by Powers (1917), who concluded from a study of manytoxic substances to goldfish that the shape of the concentration-survival time curveresembles a rectangular hyperbola, and that with most poisons there is a range ofconcentrations where the relation between concentration and 100/survival time islinear, but if the data are sufficiently extensive the points usually depart from thestraight line at the higher and lower concentrations. Our data from Exp. 3 areplotted in this way in Fig. 4, except that 100/median survival time has been usedin place of 100/time taken to overturn each fish. The points lie on a sigmoid curvesimilar to that found by Powers with most of the poisons he investigated, and, inaccordance with his practice, the dotted straight line is fitted to the central partof the curve where the slope is steepest. Powers was exploring the possibility ofusing the survival times of fish in toxic solutions to assay an unknown concentrationof poison by comparing its toxicity with those of known concentrations, and didnot investigate the departures from the straight line in any detail, but this methodhas often been used by other authors to interpret the results of toxicity tests designedto assess the danger to fish of a poison to be discharged into a river. Often the onlyconcentrations tested are within the range for which the linear relation holds, andif the straight line is extrapolated it cuts the concentration axis. Such extrapolationcan be very misleading; if there had been no data for our five lowest concentrationsit would appear that rainbow trout should not die in concentrations of less than


014 part cyanide per million, which is not the case. The method also discards anyinformation the experiment may give about the effect of concentrations below thepoint of intersection, although information on the more protracted survival timesin low concentrations is important in toxicity studies with fish, as a continuouspollution which will kill fish in several weeks could be as harmful to a fishery asone which would kill them in a few minutes or hours.

40

£ 30

20

I 10

1/

1

1/'/

f 025 0-5O14p p m CN

1-0

Cyanide parts per million

1-5 2-0

Fig. 4. Relation between concentration of cyanide and reaction time of fish plotted according tothe method of Powers (1917).

Equation (2), however, describes the relation between cyanide concentration andmedian survival time both for the lower concentrations for which concentrationappears to be linearly related to 100/survival time, and for the still lower concen-trations we have tested; it does not imply the existence of a threshold toxic con-centration.

When the logarithms of the survival times of fish in one concentration of poisonare normally distributed the expected survival time of any percentage P is given by

Expected survival time of P % = antilog {log T+ o(y — 5)},* (3)

where log T is the mean of the logarithms of the survival time, a is the standarddeviation of the logarithms of the survival times, and y is the probit correspondingto percentage P from Emmens (1948, Table 14.3).

The variances from which the standard deviations of the log survival times in• This equation is derived from that given by Bliss (1937).


concentrations between 0-25 and 0-07 part CN per million are derived differsignificantly from one another but show no systematic increase or decrease withtheir means. Had they been homogeneous, a better estimate of standard deviationover this range could have been made from their mean, than by calculation fromthe survival times in any one concentration; 5 could be substituted for a, and(log k — n log C) for log T in equation (3) to give

Expected survival time of P % at cyanide concentration C

a is calculated from the equation

S (log 7\ — log J!j)2

where Ti is the survival time of each fish in the rth test, and Nt is the number ofindividuals in the rth test.

Since the variances are heterogeneous, a value for a calculated from the data ofExp. 3 is not the best estimate of a 'true' population standard deviation commonto all the tests, but an intermediate value of significantly different standard devia-tions, no one of which is more likely to be representative of the whole series thanany other. Equation (4) has been used to calculate the lines relating concentrationand the survival times of 25 and 75% of the test fish; these are shown in Fig. 5and the times observed during the tests at which these percentages were survivingare plotted for comparison. The general agreement between the data and equa-tion (4) suggests that if it were possible to ensure identical conditions apart fromcyanide concentration in all tests and identical environmental histories for all thebatches of fish, departures from equation (4) might be due only to sampling thefish at random. If this is so, 'better standardization of the environments in whichthe batches are kept before and during a test, and the performance of tests in allconcentrations at the same time should give results in closer agreement with thehypothesis.

In biological assays where the survival times of test animals are used thelogarithms of survival time and the logarithm of dose seem more often to belinearly related than other functions of time and dose (Emmens, 1948, p. 169).Ostwald (1907) applied the relation CnT=k to data obtained by studying thesurvival of Gammarus in different dilutions of sea water. Chick (1908) found thatthis relation held for the concentration of several disinfectants and the time takento kill bacteria. Perry (1950) found that the dose of neoarsphenamine and thesurvival time of mice followed this law. Ostwald's equation seems applicable tothe effect of many drugs and poisons on a great variety of living organisms, andit appears to be true for the effect of many poisons on fish. Although Powers usedthe 1001T method to interpret his results, his data for the toxicity of substances togoldfish are well fitted by Ostwald's equation in fourteen of the sixteen cases wehave examined, and there is no systematic departure from the law at the lowerconcentrations, although in some the points depart from the line at the higherconcentrations similarly to the points along B-Bl in Fig. 3.


10.000

1,000

e^ 100

10

,25%

75%\

\

\

\

75%

1005 0-1 0-2

Parts CN per million (logarithmic icale)

Fig. 5. Relation between concentration of cyanide and time taken to overturn 25 and 75 % ofrainbow trout at 17-5° C. plotted on logarithmic scales. Continuous lines computed fromequation (4); the short vertical lines join the times at which 25 and 75 % of the fish wereobserved to overturn during the tests.

SUMMARY

1. An apparatus is described in which fifty yearling rainbow trout can be testedin a stream of water at a constant temperature and containing a constant concen-tration of poison.

2. Exposure to cyanide causes the fish to lose equilibrium control and to turnover. Resistance is measured as survival time, by which is meant the time takento overturn the fish.

3. When the experimental temperature is higher than that of the water in thestock aquaria the resistance of yearling rainbow trout to cyanide increases withincreasing time of acclimatization to the temperature of the experiment.

4. Small yearling rainbow trout tend to be more resistant to cyanide than largerfish of the same age.

5. The distribution of the logarithms of the survival times of the test fish incyanide solutions, though not precisely normal, is sufficiently close to a normaldistribution to justify the application of standard statistical techniques. Neitherthe distribution of the survival times nor that of the reciprocals of the times providea suitable basis.


6. At 17-5° C. in the range 2-5 parts to 0-07 part CN per million it has beenfound that (i) mean log survival time decreases linearly with log concentration,(ii) the variance of log survival time is approximately constant, and (iii) the distribu-tion of log survival time is approximately normal. At higher concentrations theseconclusions are not true.

7. The resistance of an individual rainbow trout to cyanide is mainly determinedby inherent properties which persist for at least three weeks.

We wish to record our thanks to the Director of Building Research for assistancefrom his statistical staff at various stages of the work. We also wish to thankMr P. S. Hewlett of the Pest Infestation Laboratory for reading the paper intypescript and for many helpful suggestions.

The paper is published by permission of the Department of Scientific andIndustrial Research.

REFERENCESAI.I.BH, W. C. & BOWEN, E. (193a). Studies in animal aggregations. Mass protection against

colloidal silver among goldfishes. J. Exp. Zool. 61, 185.BLISS, C. I. (1937). The calculation of the time-mortality curve. Ann. Appl. Biol. 34, 813.CAWHNTBR, K. (1927). The lethal action of soluble metallic salts on fishes. Brit. J. Exp. Biol. 4,

378.CHICK, H. (1908). An investigation of the laws of disinfection. J. Hyg., Camb., 8, 92.EMMKNB, C. W. (1948). Principles of Biological Assay. London: Chapman and Hall Ltd.ERICHSEN JONES, J. R. (1938). The relative toxicity of salts of lead, zinc, and copper to the stickleback

(Gasterosteus aculeatus L.) and the effect of calcium on the toxicity of lead and line salts.J. Exp. Biol. 15, 394.

GBABY, R. C. & PEARSON, E. S. (1938). Tests of Normality. Biometrika Office, University College,London.

OSTWALD, W. (1907). Uber die Beziehungen zwischen Adsorption und Giftigkeit von Salzldsungenfiir SUsswassertiere (Gammarus). Arch. ges. Pkysiol. 130, 19.

PERRY, W. L. M. (1950). Reports on biological standards. VI. The design of toxicity tests. Spec.Rep. Ser. Med. Res. Coun., Lond., no. 270. London: H.M. Stationery Office.

PETERS, C. C. & VAN VOORHIS, W. R. (1940). Statistical Procedures and their Mathematical Bases.New York: McGraw-Hill Book Co.

POWERS, E. B. (1917). The goldfish as a test animal in the study of toxicity. Illinois Biol. Monogr. 4,no. 2, 1-73.

ROBBIE, W. A. (1944). An improved phenolphthalein technique for the micro-determination ofcyanide. Arch. Biochem. 5, 49.

SUMNER, F. B. & WELLS, N. A. (1935). Some relations between respiratory metabolism in fishesand susceptibility to certain anesthetics and lethal agents. Biol. Bull. Woods Hole, 69, 368.

WUHRMANN, K. & WOKER, H. (1948). Beitrflge zur Toxikologie der Fische. II. ExperimentelleUntersuchungen liber die Ammoniak- und Blausfiurevergiftung. Schweiz. Z. Hydrol. 11, 210.

THE TOXICITY OF POTASSIUM CYANIDE TO TROUT

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